R E V I E W
Open Access
Immunotherapy in HER2-positive breast
cancer: state of the art and future
perspectives
E. Krasniqi
1, G. Barchiesi
1, L. Pizzuti
1, M. Mazzotta
2, A. Venuti
3, M. Maugeri-Saccà
1, G. Sanguineti
4, G. Massimiani
1,
D. Sergi
1, S. Carpano
1, P. Marchetti
2,5, S. Tomao
6, T. Gamucci
7, R. De Maria
8,9, F. Tomao
10, C. Natoli
11, N. Tinari
11,
G. Ciliberto
12, M. Barba
1*and P. Vici
1Abstract
Breast cancer (BC) is a complex disease with primary or acquired incurability characteristics in a significant part of patients. Immunotherapeutical agents represent an emerging option for breast cancer treatment, including the human epidermal growth factor 2 positive (HER2+) subtype. The immune system holds the ability to spontaneously implement a defensive response against HER2+ BC cells through complex mechanisms which can be exploited to modulate this response for obtaining a clinical benefit. Initial immune system modulating strategies consisted mostly in vaccine therapies, which are still being investigated and improved. However, the entrance of trastuzumab into the scenery of HER2+ BC treatment was the real game changing event, which embodied a dominant immune-mediated mechanism. More recently, the advent of the immune checkpoint inhibitors has caused a new paradigm shift for immuno-oncology, with promising initial results also for HER2+ BC. Breast cancer has been traditionally considered poorly immunogenic, being characterized by relatively low tumor mutation burden (TMB). Nevertheless, recent evidence has revealed high tumor infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in a considerable proportion of HER2+ BC patients. This may translate into a higher potential to elicit anti-cancer response and, therefore, wider possibilities for the use and implementation of immunotherapy in this subset of BC patients. We are herein presenting and critically discussing the most representative evidence concerning immunotherapy in HER2+ BC cancer, both singularly and in combination with therapeutic agents acting throughout HER2-block, immune checkpoint inhibition and anti-cancer vaccines. The reader will be also provided with hints concerning potential future projection of the most promising immutherapeutic agents and approaches for the disease of interest.
Keywords: Metastatic, Early, Breast cancer, Immunotherapy, Vaccine, HER2+
Background
The most relevant predictors of prognosis and treatment outcomes in BC include the expression of hormone receptors (HR) and the overexpression of HER2 or amp-lification of the inherent gene [1]. Breast cancer is classi-fied on the basis of the presence or absence of these factors into four intrinsic molecular subtypes: luminal A, luminal B, HER2 enriched, and triple negative breast cancer [2], which determine different biological behaviors
and diverse clinical evolutions [3]. HER2+ BC constitutes 15–20% of newly diagnosed invasive breast carcinomas [4]. HER2-blocking therapies, such as trastuzumab and/or pertuzumab in combination with chemotherapy represent the standard first-line treatment for HER2+ metastatic (m) BC [5]. In addition, several HER2-targeting therapeutics, including the drug-antibody conjugate ado-trastuzumab emtansine (T-DM1), and, less recently, lapatinib, a revers-ible tyrosine kinase inhibitor (TKI), have been approved for the treatment of this tumor [6,7]. However, HER2+ mBC will eventually progress in most patients because of primary or secondary resistance to anti-HER2-directed therapies, including trastuzumab [8]. The impellent necessity for the © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence:maddalena.barba@gmail.com
1Division of Medical Oncology 2, IRCCS Regina Elena National Cancer Institute, Via Elio Chianesi, 53-00144 Rome, Italy
development of novel therapies and new approaches to overcome the limitations of targeted therapy and improve treatment outcomes oriented our choice on the topic of interest. Indeed, immunotherapy may represent an add-itional option for HER2+ BC patients, although, thus far, the expansion of the immune-oncology field has found a relatively narrow space in the landscape of breast cancer treatment. When globally considered, breast carcinoma is classified as a moderately immunogenic cancer [9, 10]. However, studies on somatic mutations and tumor micro-environment wherein data analyses are performed by molecular subtypes have shown remarkable heterogeneity [11–13], with the highest immunogenic potential being as-cribed to triple negative breast cancer (TNBC) and HER2+ BC among BC subtypes [14]. Considering the efficacy of immunotherapeuticals in highly mutated tumors with high infiltration of immune cells, a relevant benefit from im-munotherapy is expected in the HER2+ mBC setting. The inhibition of the hyperactive HER2 protein kinase receptor by trastuzumab in HER2+ breast cancer cells [15] can be partly considered an immunotherapy strategy, since the monoclonal antibody (mAb) mechanism of action includes an immune-mediated component [16]. On this basis, clin-ical trials have addressed the possibility of enhancing this immune mechanism to overcome the anti-HER2 resistance. In the same optics of exploiting the immune-mediated mechanism of some HER2-blockers, preclinical studies have investigated their potential synergistic effect in associ-ation with the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) block or with the programmed cell death protein 1 (PD1) inhibition, demonstrating a robust lymphocytic ac-tivation against BC cells [17,18]. Currently, these combina-tions are being evaluated in different BC settings. Further promising strategies to capitalize on the immunogenic properties of HER2+ BC include the anti-HER2 vaccine therapies, which have been tested in different formulations in both preclinical and clinical studies.
Herein, we discuss preclinical evidence on mechanistic aspects potential strategies and data from clinical trials on immunotherapy for HER2+ BC. Future perspectives are also envisioned concerning the use of immuthera-peutic agents and vaccines in HER2+ BC. The inherent pitfalls and caveats of the most attractive strategies will be also briefly discussed.
Methods
We searched PubMed from inception to April 2019. We aimed at identifying both intervention trials and observa-tional studies focused on immunotherapy for HER2+ breast cancer. Intervention trials were included inde-pendently on the phase and randomized allocation. When considering observational studies, those with a prospective design and a control group were judged suit-able for inclusion. Mixed design studies, i.e., observational
studies carried out in patients enrolled in intervention trials, were allowed. Evidence from preclinical studies was also considered. References were identified by combining the following terms used both as text words and med-ical subject headings: immunotherapy, vaccines, HER2-positive, and breast cancer. Once the manuscripts of interest were identified, the reference lists were screened for additional relevant papers. In addition, we consulted the ASCO proceedings from 2010 to 2018.
Anti-cancer immune response in HER2+ breast cancer and its correlates
The development of an immune response against cancer undergoes different steps broadly encompassed within two major domains, i.e., the early phase, wherein key actors of the immune system are directly involved at the immune system organs’ level, and the late phase, that occurs at the tumor cell site and contemplates the effect-ive anti-tumor response, which contemplates a dynamic interaction with the microenvironment. In the early phase, dendritic cells (DCs) sample tumor-associated an-tigens (TAAs), such as the HER2 protein [19], and then process and present them after an appropriate matur-ation signal; otherwise, tolerance will be established [20]. An activated mature DC will then dispatch its role as an antigen presenting cell (APC) generating a T cell re-sponse, which includes the production of anti-HER2 CD8+ cytotoxic T cells and CD4+ T cells [21]. The anti-HER2 CD4+ T cells will in turn activate a specific B cell response with consequent anti-HER2 antibody produc-tion. The second phase of the immune response occurs in the peripheral area, where cancer-specific T-cells and/ or antibodies recognize HER2+ cancer cells and execute their cytotoxic effect (Fig.1).
However, as soon as cancer-specific T cells enter the tumor microenvironment, the challenge of cancer-induced immune suppression begins. Tumors can obstacle DC mat-uration, trigger an inappropriate immune response such as TAA-tolerance, or facilitate the infiltration and expansion of T regulatory cells (Tregs), which correlates with poor prognosis [22, 23]. Normally, T cytotoxic activity is finely modulated by facilitating and inhibiting signals. In more detail, after binding of the T cell receptor (TCR) to the major histocompatibility complex (MHC)-antigen complex, the B7 protein on the APC binds to the T cell costimula-tory molecule CD28 to promote T cell activation and sur-vival. Inhibition of T cell activation is mediated by the subsequent upregulation of cytotoxic T lymphocyte antigen 4 (CTLA-4) on T cells, which competes with CD28 to bind B7. Further control of T cell response in peripheral tissues is regulated by the expression of PD-1 on the activated T cells. The binding of PD-1 to its programmed death-ligand 1 (PD-L1) induces an inhibitory signal that limits T cell proliferation and cell survival [24–26]. Cancer cells may
overexpress surface molecules that cause T cell anergy, such as PD-L1 [27]. The spontaneous immune response to HER2+ BC may be eluded at any step of the processes in-volved in cancer initiation, progression, and metastatic spread. Therefore, the development of a successful cancer immunotherapy strategy in HER2+ BC has to contemplate the overcoming of all the above barriers. In accordance with the aforementioned two domains of immune response, we can divide the immunotherapeutic strategies into two groups: the first one seeks to enhance the maturation/activ-ity of the DCs/T effector cells, and can be performed throughout the use of vaccines; the second one comprises all the modalities developed to overcome the immunosup-pressive mechanisms at the tumor microenvironment level and can be mediated by immune checkpoint inhibitors and partially by enhancing the antibody-dependent
cell-mediated citotoxicity (ADCC) effect of the anti-HER2 agents.
When considering anti-cancer immunotherapeutic strategies for HER2+ BC, it is important to define the in-herent immunogenicity, which has some proposed sur-rogates such as tumor mutational burden (TMB), tumor infiltrating lymphocytes (TILs), and PD-L1 expression. Whole exome sequencing of tumor samples has uncov-ered a positive association in melanoma and non-small cell lung cancer (NSCLC) between response to immuno-therapy and TMB [28,29]. Higher TMB is also predictive of better clinical activity of immunotherapy in terms of objective response (OR), progression-free survival (PFS), and overall survival (OS) in colorectal cancer [30,31] and other tumor types [29,32,33]. The median somatic muta-tion frequency of BC is 10 times lower than that of highly
Fig. 1 Graphic representation of anti-cancer immune response in HER2+ breast cancer. The anti-cancer immune response is composed by two phases: in the first phase (early phase), DCs sample, process, and present TAAs (such as HER2) generating T cell triggering (both CD4 and CD8) with consequent specific antibody production and cytotoxic cell activation. The process involved in the first phase is responsible for the development of vaccines. The second phase takes place in peripheral areas where specific anti-HER2 antibodies and activated cytotoxic cells explicit their functions. The second phase is the main target of other anti-tumor drugs such as anti-HER2 antibody, antibody-drug conjugate, tyrosine kinase inhibitors, or immune checkpoint inhibitors. Legend: Ab-HER2= anti-HER2 antibody; APC= antigen-presenting cell; B7= B7 protein; CD28= T cell costimulatory molecule CD28; CTLA-4= cytotoxic T-lymphocyte–associated antigen 4; DC= dendritic cell; HER2= human epidermal growth factor 2; MHC= major histocompatibility complex; PD-1= Programmed cell death protein 1; PDL-1= Programmed death-ligand 1; TAAs= tumor-associated antigens; TCR= T-cell receptor; Tregs= regulatory T cells; T CD8= CD8+ cytotoxic T cell; T CD4=CD4+ T cell
immunogenic tumors such as melanoma, NSCLC, bladder cancer, and colorectal cancer [9, 34]. However, there is high variability among BC subtypes, especially between hormone receptor positive (HR+) tumors and the hor-mone negative (HR-) ones, with these latter generally showing higher TMB [35,36]. Also HER2 positivity in BC confers higher TMB, as a study on a HER2+ mBC patient population showed. More than one third of tumors ana-lyzed in the study had a high TMB (> 100 mutations), which predicted a longer median overall survival (mOS) (p = 0.016) [37].
The second immunogenicity correlate is represented by the number of immune cells infiltrating breast cancer bed—tumor infiltrating lymphocytes (TILs), expression of a specific pre-constituted immunity. Tumors with more than 50–60% of TILs, defined as lymphocyte-predominant BC (LPBC), have relatively good prognosis even if they belong to a less favorable subtype such as TNBC or HER2+ [38]. Incidence of LPBC is 20% for TNBC, 16% for HER2 subtype, and 6% for ER-positive luminal subtype [39]. TNBC and HER2+ tumor patients with more TILs have also better outcomes with standard treatment in terms of response to neoadjuvant chemo-therapy, event-free survival (EFS), and OS [38–47]. This evidence suggests potentiality of TILs as a predictive and prognostic marker in early/locally advanced HER2+ BC.
The third correlate, which is currently used as a pre-dictive biomarker for immunotherapy in some tumors, is PD-L1 expression. The programmed death-ligand 1 (PD-L1) antigen on tumor cells (and some immune cells) interacts with PD-1 on the immune cell surface to inhibit cytotoxic T-cell activity and stimulate regulatory T-cell development, thereby extinguishing the immune response [48]. The PD-L1 expression is heterogeneous across breast cancers and is generally positively associ-ated with the presence of TILs and aggressive molecular subtypes such as TNBC and HER2+ BC [49]. Studies including mixed populations of patients with BC in relation to the tumor subtype (mostly TNBC in the early setting), showed that higher PD-L1 expression in BC is gen-erally associated with longer OS in early disease [50–54], higher pCR rate after neoadjuvant treatment and better EFS [53, 55, 56]. A study conducted in a pure cohort of HER2+ BC patients with locally advanced disease showed an 18% frequency of PD-L1 expression, which was posi-tively associated with high TILs and longer OS [57]. Data suggest that PD-L1 expression may be considered a rele-vant prognostic and predictive factor in HER2+ BC, whose role needs to be better defined. In the following sections, evidence on anti-cancer immune response was classified by prioritizing the treatment strategy (HER2-block, immune checkpoint inhibition, anti-cancer vaccines). As it will be reflected in the heading of each respective section, evidence on HER2-block regards strategies to improve its
immune-mediated mechanism, evidence on immune checkpoint in-hibition is focused mostly on the advanced disease, while data on anti-cancer vaccines were more abundant for the early disease.
Enhancing the HER2-block immune-mediated mechanism
In invasive HER2+ BC, trastuzumab represents a land-mark of treatment, which has been confirmed to im-prove clinical outcomes across the different settings [58–60]. It is a humanized antibody that blocks HER2 signaling inducing G1 cell-cycle arrest and inhibition of PI3K/Akt pathways, which lead to apoptosis and inhib-ition of angiogenesis [61]. Besides this anti-trophic effect, evidence has sustained also an immune-based mechanism of trastuzumab. Throughout a human fractal crystallizable (Fc) region, trastuzumab is able to facilitate ADCC and prime target cells for attack by the immune system [62,63]. Trastuzumab treatment is characterized by the occurrence of primary or secondary resistance in virtually all patients with metastatic disease [64]. Subsequent anti-HER2 agents have been developed in order to extend the efficacy of HER2-block beyond the resistance obstacle.
Newer agents such as lapatinib, a dual HER1/HER2 kinase inhibitor, the HER2/HER3 dimerization inhibitor pertuzumab and more recently, the panher (HER1, 2, and 4) kinase inhibitor Neratinib can postpone or overcome anti-HER2 resistance and have yielded clin-ical advantages when administered in combination with chemotherapy, hormone therapy, and/or another HER2 inhibiting agent [5,6,65]. However, pertuzumab improves only the anti-trophic effect of the HER2-block, while as it was shown by the EGF104900 study, lapatinib also amplifies the trastuzumab-induced ADCC effect [66]. Other agents or strategies have been developed in order to enhance the immune mechanism revealed
during trastuzumab-mediated HER2-block (Table 1).
Ado-trastuzumabemtansine (T-DM1) is an antibody-drug conjugate, composed of trastuzumab and the cytotoxic component DM1, which increases the thera-peutic range of the anti-HER2 mechanism by partially overcoming the anti-HER2 resistance to other HER2-blockers. The cytotoxic component DM1 acts by a microtubule depolymerizing mechanism, which has been shown to facilitate the DC functions [7, 85–89]. Preclinical studies have investigated a possible role of T-DM1 as a potential synergic factor in combination with immune checkpoint inhibitors like anti-PD-L1 or anti-CTLA-4 [17, 18]. Proceeding from these results, the KATE II study evaluated the combination of the anti-PD-L1 atezolizumab with T-DM1, showing prom-ising preliminary results in previously treated HER2+ mBC in terms of response rate and progression-free survival (PFS) [90].
Table 1 Published clinical trials of me tastatic HER2 + breast cancer patients treated with immunotherapy Name Drug Phase Setting Number of HER2-positive patients Type of intervention Mechanism of action Outcomes Diéras et al. [ 7 ] TDM-1 III Metastatic 991 HER2+ block enhancer Antibody conjugate -PFS (p< 0.001) -OS (p< 0.001) Bang et al. [ 67 ] Margetuximab I Metastatic 27 HER2+ block enhancer ADCC stimulation against HER2 receptor -Dose finding -Clinical benefit rate Alsina et al. [ 68 ] MCLA-128 I/II Metastatic 8 HER2+ block enhancer ADCC stimulation against HER2/HER3 receptors -Dose finding -Clinical benefit rate Borghaei et al. [ 69 ] 2B1 I/II Metastatic 20 HER2+ block enhancer NK stimulation against HER2 receptor -Objective response rate -OS -TTP Dirix et al. [ 70 ] Avelumab Ib Metastatic 26 Immune check point inhibitor Anti PD-L1 -Objective response rate -PFS -OS Manguso et al. [ 71 ] Pembrolizumab Ib/II Metastatic 58 Immune check point inhibitor Anti PD-1 -Disease control (PD-L1 neg) -PFS -OS Disis et al. [ 72 ] NA I/II Metastatic 22 Vaccine Peptide-based vaccine Immunological response Curigliano et al. [ 73 ] NA I/II Metastatic 40 Vaccine Recombinant HER2 protein + immunostimulant -PFS -Objective response rate Hamilton et al. [ 74 ] NA I Metastatic 12 Vaccine Recombinant HER2 protein + immunostimulant + Lapatinib Immunological response Miles et al [ 75 ] NA III Metastatic 157 (15% of total patients) Vaccine Mucine-epithope (STn-KLH) Overall TTP Overall OS (p = 0.916) Heery et al. [ 76 ] PANVAC II Metastatic 6 (12% of total patients) Vaccine DNA-based vaccine Overall PFS (p = 0.09) Norell et al. [ 77 ] NA I Metastatic 8 Vaccine Plasmid vector-based vaccine Immunological response Tiriveedhi et al. [ 78 ] NA I Metastatic 3 (21% of total patients) Vaccine DNA-based vaccine -Immunological response -Overall PFS (p = 0.011) Morse et al. [ 79 ] NA I Adjuvant/ Metastatic 7 Vaccine Dendritic cell vaccine -Immunological response -DFS Emens et al. [ 80 ] NA I Metastatic 1 (0.03%of total patients) Vaccine Anti-HER2 allogenic breast cancer cell line Immunological response Dols et al. [ 81 ] NA I Metastatic 12 (43% of total patients) Vaccine Allogenic breast cancer cell lines -Immunological response -TTP -OS Chen et al. [ 82 ] NA Metastatic 22 Vaccine Granulocyte macrophage colony-stimulating factor (GM-CSF) –secreting tumor vaccine -Immunological response -Objective response rate -PFS -OS Park et al. [ 83 ] Lapuleucel-T I Metastatic 19 Vaccine Dendritic cell based vaccine -Immunological response -Objective response rate Disis et al. [ 84 ] NA I Metastatic 7 Vaccine Autologous T-cell vaccine -Immunological response -Objective response rate Abbreviations: ADCC antibody-dependent cytotoxic cell, NK natural killer, NA not available, OS overall survival, PFS progression-free survival, TTP time to progression
Another strategy for exploiting the immune-mediated anti-cancer activity of anti-HER2 agents has been the optimization of their Fc in such a way that it becomes more efficacious in activating the ADCC. Margetuximab is a new generation mAb that targets the HER2 pathway and has a Fc region with an increased ability to mediate ADCC executed by effector cells such as NK cells and monocytes. A phase I trial tested this mAb in heavily pretreated HER2+ mBC patients, showing good tolerabil-ity and activtolerabil-ity in this setting of patients [67]. The primary analysis of the SOPHIA trial, a randomized phase III trial comparing margetuximab plus chemotherapy versus tras-tuzumab plus chemotherapy in patients with HER2 + mBC who received a maximum of three prior lines was re-cently presented at the ASCO symposium. Margetuximab plus chemotherapy improved PFS (5.8 months versus 4.9, p = 0.033), ORR, and clinical benefit rate (CBR) compared with trastuzumab plus chemotherapy with an acceptable safety profile, similar to trastuzumab [91]. MCLA-128 is another anti-HER2 mAb developed as a bispecific IgG1 to enhance ADCC activity and overcome HER3-mediated resistance developed under classic HER2 blockers. A phase I/II study showed a good safety profile and consist-ent anti-tumor activity in heavily pretreated mBC paticonsist-ents who developed disease progression while on anti-HER2 therapies [68].
Other anti-HER2 bispecific antibodies have been de-veloped under the optics of enhancing immune response to HER2+ cancer cells by promoting the encounter be-tween lymphocytes and HER2+ cancer cells. A preclin-ical study investigated an engineered bispecific antibody for HER2 and CD16, employed to redirect CD16-expressing T lymphocytes and NK cells towards HER2+ cancer cells. The inherent results revealed the superiority of this agent compared to trastuzumab in triggering im-mune response [92]. A phase I/II clinical trial employed this bispecific antibody for treatment of patients with HER2+ mBC. Objective anti-tumor responses were not obtained. However, the therapy induced adaptive immune responses to both the HER2 intra-cellular domain (ICD) and extra-cellular domain (ECD) [69]. CD137 and CD3 are two T cell specific costimulatory receptors, which promote cell proliferation, survival, and activation [93]. The agonistic engagement of these receptors by bispecific mAbs which also bind to HER2 could both facilitate the encounter between HER2+ cancer cells and T lympho-cytes and stimulate the activation of these latter [94]. PRS-343 is a bispecific fusion protein bridging CD137+ T cells with HER2+ tumor cells physically and functionally [95]. It is being tested in phase I trials for HER2+ solid tumors including breast cancer as a monotherapy [96]. Preclinical studies have suggested that the immunostimulatory ap-proaches, such as agonistic CD137 mAb and anti-PD-L1 mAb therapy, may be used to capitalize on the
immune-mediated effects of trastuzumab [17]. There is an ongoing trial exploring the feasibility of treating HER2+ BC with a combination of PRS-343 and the anti-PD-L1 atezolizumab [97]. An additional ongoing phase I trial is evaluating a HER2-directed bispecific antibody, which binds to CD3 in order to engage T cells against HER2+ cancer cells in different HER2+ solid tumors, including BC [98]. Another possibility that has been explored is re-lated to the use of nanobody activation immunotherapeu-tics against the HER2 protein. A preclinical study focused on the efficacy of a nanobody agent that selectively redi-rects anti-dinitrophenyl (DNP) antibodies to the surface of HER2+ BC cells, resulting in their targeted destruction by ADCC. This study showed that this agent is capable of se-lectively binding to HER2+ BC cells, recruiting anti-DNP antibodies to the cell surface, and activating ADCC [99].
An additional immune mechanism of trastuzumab that may be fostered for therapeutic purposes could theoret-ically be its ability to increase tumor infiltration by lymphoid cells [16]. The increased recruitment of TILs and their intra-tumoral expansion is expected with the combination of an IL-2 variant targeting fibroblast acti-vation protein-alpha and trastuzumab in an ongoing phase I trial [100] (see also Tables3and4).
Also other preclinical studies suggest possible combin-ation of anti-HER2 therapy with cytokines. A study showed that a combination of interferon gamma (IFN-γ) and anti-HER2 antibody synergistically reduce tumor growth in mammary tumor models [130]. On this basis, a small study aimed at using a recombinant approach to produce an anti-HER2 single-chain variable domain fragment (scFv) and IFN-γ fusion protein, which demon-strated superior activity over the anti-HER2 antibody and was even active on tumors that were resistant to anti-HER2 antibody therapy [131].
A further strategy that has been explored to improve trastuzumab anti-cancer efficacy is labeling it with a radionuclide. A pilot study evaluated the feasibility of treating HER2+ mBC patients refractory to previous therapies with radioimmunotherapy developed by attach-ing the radioactive lutetium-177 (Lu-177) to trastuzumab. This study showed that the treatment was feasible and safe and could be considered for palliative treatment of HER2+ mBC in combination with standard agents [132].
Finally, besides the development of resistance, trastu-zumab presents pharmacokinetic limitations because the reaching of a therapeutic concentration at the tumor site is often hampered by potential toxicities [133]. Preclinical studies have explored strategies to overcome this barrier. A cancer-selective oncolytic adenovirus was engineered to encode trastuzumab antibody chains allowing the produc-tion of monoclonal anti-HER2 antibody directly by cancer cells, which are then lysed, releasing both new virions and the Tumor-associated antigens (TAAs) for dendritic cells
(DC) recognition and activation. Efficacy of this strategy in HER2-+ cancer was shown in vivo [134,135]. Another in vitro study reported an efficient antibody delivery sys-tem for the incorporation of trastuzumab into poly (lactic-co-glycolic) acid nanoparticles (PLGA NPs) to overcome poor pharmacokinetics and low tumor penetration by the monoclonal antibody [136].
Immune checkpoint inhibitors in advanced disease
One of the most important breakthroughs in cancer im-munotherapy has been recently reached with the advent of the immune checkpoint inhibitors, which confer to cancer patients a clear survival advantage. Although ini-tial steps have been explored with immune checkpoint inhibitors in BC, significant results are still lagging be-hind in HER2+ disease. However, there seems to be a strong rationale to move forward also in this direction, since the studies show that HER2+ BC is characterized by intrinsic immunogenicity. Moreover, immunotherapy is already exploited in a very effective way in HER2+ BC, since the predominant mechanism of trastuzumab is im-mune mediated. Clinical studies showed that higher TILs could have prognostic and predictive potential in HER2+ BC, besides the evidence of synergy with trastu-zumab [38, 40, 137], which may indicate a synergic ac-tion between HER2-directed therapy and checkpoint inhibition. This has been the basis leading to the com-bination strategies. The anti-PD-1/PD-L1 agents act at the effector stage by re-energizing pre-existing T cells, while anti-CTL-4 agents act at the proliferation/activa-tion stage by also probably enhancing de novo re-sponses. A list of the most recent trials of immune checkpoint inhibitors administered in association with each other or combined with standard therapies or other immunomodulating strategies, e.g., vaccines, in HER2+ BC is shown in Table1.
The inhibition of the PD-1/PD-L1 synapse at the per-iphery level has yielded promising outcomes. The pro-grammed cell death ligand 1 can be expressed by both tumor cells and immune cells and in BC its expression correlates with hormone receptor negativity, higher histo-logical grade, and higher TILs [138]. Most of the data on anti-PD-1/PD-L1 inhibitors in BC derive from studies carried out in TNBC patients. Several early-phase studies investigating the PD-1 inhibitor pembrolizumab and the PD-L1 inhibitors atezolizumab and avelumab as mono-therapy for mTNBC have reported objective response rates (ORRs) ranging from 4 to 23% and superior out-comes in the first line and in patients having tumors that expressed PD-L1 in ≥ 1% of cells [70, 139–141]. Associ-ation with chemotherapical drugs such as nab-paclitaxel and eribuline increased the efficacy of atezolizumab in terms of ORRs in mTNBC [142,143]. In these same set of patients, the combination of atezolizumab with
nab-paclitaxel yielded also an OS advantage in the phase III trial Impassion 130, [144], while the anti-PD1 pembrolizu-mab in association with standard neoadjuvant chemother-apy raised the pCR rate to 60% and 80% in two different studies [145,146]. Considering that HER2+ BC and TNBC share similarities in terms of immunogenicity correlates such as tumor mutation burden (TMB), TILs and PD-L1 expression, a significant benefit from immune checkpoint inhibitors is expected also in HER2 overexpressing BC. However, data in this setting are relatively scarce and no single agent-study has been conducted in a pure cohort of HER2+ BC patients. As we described previously, the tras-tuzumab mechanism of action contemplates also an immune-mediated action and synergic effect with the combination of checkpoint with HER2-directed therapy has been observed in preclinical studies [17,18,147].
One of the major concerns regarding anti PD/PDL1 therapies is acquired resistance. Several underlying mecha-nisms have been suggested. One of the most studied pro-cesses involves the JAK1/2 signaling: interferon-gamma (IFN)-JAK1/2-STAT1 pathway, which plays a key role in T-cell-mediated citotoxicity. Evidence in support of a role of IFN gamma deregulation has come from studies of next-generation sequencing (NGS) assessment in melanoma pa-tients treated with anti PD1 agents, wherein loss of function mutations in JAK1/2 were revealed at the time of PD in pa-tients who had been previously (NGS) evaluated and re-sulted wild type at the time of response. Functional studies showed that the inactivation of JAK2 is responsible of a lack of response to IFN-gamma, which translates into a resist-ance to T cell-mediated cytotoxicity and an advantage to the tumor cells [148]. In addition, the comparative analysis of tumor samples in another patient showed a truncating mutation of B2M, a protein involved in the transport of MHC I molecules on the cell surface. A reduced or absent MHC I expression could impair T cell recognition of tumor cells, thus favoring immune escape [148].
Findings from studies of Clustered Regularly Inter-spaced Short Palindromic Repeats (CRPSR) screening, which enables genome-wide interrogation of gene func-tion, confirmed the role of PD/PD-L1 as a mechanism of immune evasion and the impairment of IFN-gamma as acquired resistance system. Interesting clues have also emerged concerning deletion of the protein PTPN2 in tumor models, which has been associated with an in-creased efficacy of immunotherapy by enhancing IFN gamma pathway [71]. In addition, immunocompetent mice have shown remarkable depletion of Adar1-targeting single-guide RNAs. ADAR1 is a protein belonging to the ADAR family, a group of enzymes responsible for RNA editing, which have been recently explored for their role in promoting immunotherapy resistence. The inherent findings showed that loss of ADAR1 increased tumors’ sensitivity to IFN and enhanced tumor inflammation, as
shown by the massive presence of CD4+, CD8+, and NK cells [149]. A further proposed mechanism of acquired resistence is related to LNK iperexpression. LNK is a key negative regulator of JAK-STAT signaling with an import-ant role in hematological malignancies. LNK is highly expressed in melanoma cell lines. The aberrant concentra-tion of LNK confers a selective survival advantage to the cells by promoting cell growth and survival and impairing the IFN-JAK1/2-STAT1 pathway [150]. The aforemen-tioned mechanisms of acquired resistance remain in need of validation in breast cancer, especially in HER2+ BC pa-tients, for whom the pertinent literature is particularly limited. To our knowledge, only two phase I/II trials have been published thus far.
The JAVELIN study is a phase Ib trial which tested Avelumab monotherapy (a human anti-PD-L1 IgG1 mAb) in patients with pretreated mBC, unselected for PD-L1 ex-pression and breast cancer subtype. A total of 168 patients were treated, of whom 15.5% had a HER2+ disease. At a threshold of≥ 1% tumor cell staining positive for PD-L1, 62.5% of patients had PD-L1+tumors. Treatment was well tolerated but no OR was recorded in HER2+ patients. This may suggest that a previously treated population may not be suitable for the evaluation of anti-PD-L1 therapy in HER2+ BC, although the relatively restricted number of patients and limited statistical power should be considered in results’ interpretation [70].
In the phase Ib/II PANACEA study, pembrolizumab in combination with trastuzumab was explored in HER2+ mBC patients who had progressed while on tras-tuzumab. Among them, 29% had received also pertuzu-mab and 72% had received prior T-DM1. The 58 enrolled patients received concurrent trastuzumab with pembrolizumab. Six patients were enrolled in the phase Ib, they all had PD-L1+ HER2+ BC and no dose-limiting toxicities were recorded. The phase II portion of the study enrolled 52 patients, of whom 77% had PD-L1+ disease, the remaining were PD-L1- HER2+ BC patients. The median follow-up for PD-L1+ and PD-L1- patients was respectively 13.6 months and 12.2 months. Seven (15%) of the 46 PD-L1-positive patients achieved an ob-jective response, and further four (8%) patients main-tained a stable disease for more than 6 months as their best response, with disease control being recorded in 11 (23%) within the subgroup of PD-L1-positive patients. The mean duration of disease control was 11.1 months. By contrast, there were no objective responders in the PD-L1-negative group. The preliminary subgroup ana-lyses of this study showing higher levels of TILs in the PD-L1 positive tumor population, suggested that im-mune mechanisms might be important in trastuzumab resistance and selection for patients who are PD-L1-positive. In future studies of metastatic HER2+ disease testing HER-2 blockade combined with anti-PD-1 or
anti-PD-L1 drugs, the analysis of TILs is a nodal point which deserves further investigation [151].
Recently, the randomized phase II KATE2 study evalu-ated the addition of atezolizumab to T-DM1 in patients with locally advanced or metastatic HER2+ BC patients who received prior trastuzumab and taxane-based ther-apy. The primary endpoint was PFS. One hundred thirty-three patients were randomized to receive atezoli-zumab plus DM1 and 69 patients to placebo plus T-DM1. Almost half of the patients in both arms had re-ceived prior pertuzumab for metastatic BC. The primary results of the study were presented at the 2018 San Antonio Breast Cancer Symposium. Overall mPFS in months was 8.2 in the atezolizumab group vs. 6.8 in the placebo group with no statistical significance (HR = 0.82; p = 0.33). However, some other interesting outcomes were reported. In the atezolizumab group, patients with PD-L1+ disease had longer PFS compared to those with PD-L1-disease, while the opposite finding emerged from patients allocated to the placebo arm. In the
atezolizu-mab group, patients whose tumors showed TILs ≥ 5%
had longer PFS with respect to patients with TIL < 5% patients. This evidence was reversed in the placebo group. No differences in ORR emerged between the two treatment arms. When patients were compared with re-spect to PD-L1 positivity and a TIL 5% cut-off value, the same phenomena were also observed for ORR. Treat-ment was well tolerated in both arms. Atezolizumab plus T-DM1 did not yield a clinically significant PFS benefit. However, a suggestion of more favorable PFS and ORR were seen with the combination in patients with PD-L1+
and/or TIL ≥ 5% disease, who seemed to have a worse
prognosis if not treated with atezolizumab [90].
There are several ongoing trials using PD-1/PD-L1 in-hibition and/or CTLA-4 blockage in combination with standard anti-HER2 therapy for HER2+ BC. The phase II DIAmOND study is investigating the combination of PD-L1 and CTLA-4 inhibition added to trastuzumab in patients with HER2+ mBC who progressed on prior trastuzumab-based therapy. These patients receive in-duction with the PD-L1 durvalumab, then the anti-CTLA-4 tremelimumab every 4 weeks and trastuzumab. The primary endpoint is 1-year PFS [101].
The ongoing AVIATOR TBCRC045 trial is combining a CD137 agonist with an anti-PD-L1. CD137 is expressed on activated T cells and NK cells and is up-regulated by trastuzumab, which could add a synergic ef-fect to the CD137 agonist. Forty patients with advanced HER2+ PD-L1 unselected BC will be randomized to re-ceive vinorelbine, trastuzumab, and avelumab; vinorel-bine and trastuzumab; or vinorelvinorel-bine, trastuzumab, avelumab, and utomilumab, a fully human IgG2 agonist mAb that binds to the ECD of CD137. Those receiving the 2 drugs will then go on and receive the other 3
drugs, while the remaining 2 arms will discontinue ther-apy. Patients must have received prior trastuzumab and pertuzumab but no prior immunotherapy [102].
The NRG BR004 is an ongoing phase III double blind trial comparing paclitaxel, trastuzumab, and pertuzumab with or without atezolizumab in patients with metastatic HER2+ breast cancer. The primary outcome is PFS [103].
Two additional, interesting randomized phase III trials are ongoing to investigating the efficacy of HER2 double block plus PD-L1 inhibition in the neoadjuvant plus adjuvant setting for HER2+ BC PD-L1 unselected BC patients. The APTneo trial is comparing trastuzumab-pertuzumab double block plus chemotherapy to atezoli-zumab plus the double block plus a chemotherapy, which will be different for half of the patients, with respect to the non-atezolizumab cohort. Five year event-free survival is the primary endpoint [118]. The Impas-sion 050 is randomizing HER2+ BC patients with locally advanced disease to receive as a pre-operatory treatment the combination of atezolizumab plus trastuzumab-pertuzumab double block plus chemotherapy or the same treatment without atezolizumab. Pathological complete response is the primary endpoint [119] (see Tables3and4for an extend list of the ongoing trials).
Vaccines in early disease
Patients affected by cancer can spontaneously harbor specific CD8+ and CD4+ T cells for TAAs [152]. Cancer vaccines might enhance these pre-existing responses and induce de novo activation. Many initial clinical trials using cancer vaccines, which included patients with ad-vanced stages of disease, showed poor outcomes because short peptides and ineffective DC-activating adjuvants were used [153], and anti-tumor immune response might be relatively defective in metastatic cancer [154]. Subsequently, cancer vaccine development was redefined in terms of the selected TAAs and immune adjuvants and the best clinical setting in which to be exploited, by proposing the early or minimal disease stages as the most suitable scenarios [155].
Barriers to success of cancer vaccines as single peptidic agents are numerous. The TAAs bound to MHC-I on the tumor may still not be sufficiently immunogenic and expression within the tumor bed can be heterogeneous
[156]. Besides using peptides [157, 158], other
ap-proaches for vaccine development include gene vectors encoding TAAs [159] and cell-based approaches such as autologous cancer cells [160], allogenic tumor cell lines [161] and DC-based vaccines [162]. A further immuniz-ing strategy was developed by usimmuniz-ing adoptive lymphocyte therapy using lymphocytes with tumoricidal potential [163]. The possibility of successful strategies for adoptive T cell therapy beyond patients harboring pre-existing tumor-specific T cells and MHC restriction have
substantially increased with the recent advances in T cell engineering through chimeric antigen receptors (CARs) enriched by costimulatory signaling domains [164].
The anti-cancer vaccine strategy may be used to en-hance the immune response against HER2+ BC. The driver oncogene product, HER2, is expressed on the sur-face of cancer cells, where antibodies can detect it on the intact membrane. Trastuzumab’s high efficacy showed that antibodies against HER2 can yield an anti-cancer effect. However, the majority of vaccines devel-oped for HER2+ BC treatment induce T cell responses to HER2, not antibody responses [165]. Nevertheless, vaccine formulations that elicit exclusively an anti-HER2 antibody response (with no T cell response) have been developed preclinically and also tested in the (early) clin-ical setting [166]. Diverse vaccine formulations have been explored for the treatment of the BC subtypes [167]. We are now mentioning and briefly discuss evi-dence from the main clinical trials having administered cancer vaccines in HER2+ BC patients or that included also this subgroup of BC patients (Tables1and2). Peptide-based and other epitope-based vaccines
HER2 protein, carbohydrate antigens such as the carcino-embryonic antigen (CEA), and Mucin-1 (MUC-1) are the three most studied BC antigens for vaccine development. Spontaneous antibody response to HER2 and MUC-1 an-tigens in BC patients is very low. Other targets that have been explored include Wilms’ tumor 1 antigen (WT1) and gangliosides. Peptide-based vaccines have been used mostly with the granulocyte macrophage colony stimulat-ing factor (GM-CSF) cytokine as adjuvant to enhance effi-cacy [185]. They are modifiable and combinable and are associated with minimal toxicity, while offering the possi-bility for a potential prolonged immunity, but also charac-terized by HLA restriction and scarce diversity [186]. Anti-HER2 protein vaccines
Three main peptides derived from the protein structure of HER2 have been used to develop BC vaccines. The E75 and GP2 peptides, which are HLA-A2/A3-restricted, and AE37, which is a promiscuous HLA class II binder [168–170]. These peptides are located on the extra-cellular domain (E75), transmembrane portion (GP2), and intracellular domain (AE37) of the HER2 protein. However, formulations including a mixture of peptide sequences derived from both the ECD and ICD, such as the recombinant protein dHER2 [171], or other formula-tions have also been developed [72].
E75 vaccine Several phase I trials tested E75 vaccine with different immune-adjuvants in mBC with scarce anti-tumor activity [153]. However, interesting results were obtained in early BC. HER2 is expressed at different
Table 2 Published clinical trials of HER2 + breast cancer patients treated with immunotherapy, early setting Name Drug Phase Setting Numb er of HER2 -positive patie nts Type of interve ntion Mec hanism of action Outcome s Benavid es et al. [ 168 ] N A I Adjuvant 151* Vaccine Pept ide-based vac cine (E75) -Immun e respon se (p = 0. 02) -DFS (p = 0.4 LE; p = 0.7 O E) -Mortal ity (p = 0.08 LE; p = 0.6 O E) Carmic heal et al. [ 169 ] N A I Adjuvant 18 Vaccine Pept ide-based vac cine (GP2) -Safety -Immun e respon se (p = 0. 00000 4) Holm es et al. [ 170 ] N A I Adjuvant 15 Vaccine Pept ide-based vac cine (AE37) Immun ological res ponse Liment ani et al. [ 17 1 ] N A I Adjuvant 61 Vaccine Recom binant HER2 protei n + im muno stimulant Immun ological res ponse People s et al. [ 172 ] N A N A Adjuvant 53 Vaccine Pept ide-based vac cine (E75) DFS (p < 0.19 ) Mitte ndorf et al. [ 173 ] Neli pepimut-S I/II Adjuvant 187 Vaccine Pept ide-based vac cine (E75) DFS (p = 0.08 ) Patil et al. [ 174 ] N A I/II Adjuvant 52 (28% of all patients ) Vaccine Pept ide-based vac cine (E75) -Immun ological res ponse -DFS (0.5 2) People s et al. [ 175 ]; Mitte ndorf et al. [ 176 ] NA NA Adjuvant 186 Vaccine Pept ide-based vac cine (E75) -Immun ological res ponse -DFS (p = 0.04 20 month s; p = 0.08 24 mon ths) Mitte ndorf et al. [ 177 ] N A II Adjuvant 101 Vaccine Pept ide-based vac cine (GP2) DFS (p = 0.43 ) Clifton et al. [ 17 8 ] N A I Adjuvant 17 Vaccine Pept ide-based vac cine (GP2) Immun ological res ponse Mitte ndorf et al. [ 179 ] N A II Adjuvant 144 Vaccine Pept ide-based vac cine (AE37) -DFS (p = 0.45 ) -Immun ological res ponse Higgins et al. [ 180 ] NA I Neoadj uvant 15 (25% of total patie nts) Vaccine Wi lms ’tumor 1 (WM 1) im muno therapeutic -Objective response rate -Immun ogenic ity Valde s-Zayas et al. [ 181 ] N A III Adjuvant 22 (25% of total eva luated patien ts) Vaccine Anti -gangly oside vac cine (Ne uGcGM 3) Immun ological res ponse Czerniecki et al. [ 182 ] N A I Neoadj uvant 13 Vaccine Dend ritic cell bas ed vaccin e Immun ological res ponse Koski et al. [ 183 ] N A I Neoadj uvant 27 Vaccine Dend ritic cell-bas ed vac cine -Immun ological res ponse Lowe nfeld et al. [ 184 ] N A I Neoadj uvant 54 Vaccine Dend ritic cell-bas ed vac cine -Immun ological res ponse -pCR *Patients were stratified by HER2 expression and divided into low expressors and overexpressors. Low expressors were defined as HER2/neu immunohis tochemistry (IHC) 1(+) to 2(+) or fluorescence in situ hybridization < 2.0. Overexpressors were defined as IHC 3(+) or fluorescence in situ hybridization > or = 2.0 + Abbreviations: ADCC antibody-dependent cytotoxic cell, DFS disease-free survival, LE low expressors, NK natural killer, NA not available, OE overexpressors, OS overall survival, pCR pathological complete response
levels in > 75% of BC cases. A study in the adjuvant setting showed that low HER2 expressors (immunohistochemis-try 1+ (IHC1+)), generally display more robust immuno-logic responses and possibly derive the greatest clinical benefit from the E75 vaccine [168]. Different phase I and phase II trials were conducted in the adjuvant setting, mostly for high-risk BC with any level of HER2 expression (IHC 1+ to 3+) using E75 vaccine plus GM-CSF, in pa-tients who had completed standard treatment. A small study compared 24 vaccinated patients to 29 non-vaccinated ones. Disease-free survival (DFS) rates at 22 months were 85.7% and 59.8%, respectively (p = 0.19) [172]. Another phase I/II clinical trial used a similar treat-ment in this clinical setting by comparing 108 vaccinated patients to 79 controls. The DFS rates at 60 months were 89.7% and 80.2%, respectively (p = 0.08). The number of HER2+ BC patients who received trastuzumab was com-parable across the two arms and, also in this subset of pa-tients, the DFS advantage was maintained, although at a not statistically relevant extent [173]. A previous study showed that E75 vaccine plus GM-CSF were effective in raising anti-HER2 immunity in both A2+ and HLA-A3+ patients in the adjuvant setting. At a 26-month follow-up, DFS rates were 91.7% in the A2+/A3+ groups taken together and 85.2% in the control group, while at 30 months, the DFS rate was 92.3% for A3+ patients (p = 0.52). It is noteworthy that the HER2 positivity rate was significantly higher in the HLA-A2+ vaccinated group [174]. The combined results of two additional trials that investigated E75 vaccine plus GM-CSF in patients with high risk breast cancer added some interesting hints for future investigations [175]. A total of 101 HLA-A2+ and HLA-A3+ patients were vaccinated and compared to 85 HLA-A-/HLA-A2- non-vaccinated patients. The HER2+ BC patients constituted 25.8% of the vaccinated individ-uals and 28.4% of the control group. At a median follow-up of 20 months, DFS rates of the vaccinated grofollow-up vs. control group were 94.4% and 85.8%, respectively (p = 0.04). This difference ceased to be statistically significant at a 24 months median follow-up, when DFS rates were respectively 94.3% vs. 86.7% (p = 0.08). Subset analyses showed that patients benefited more from the vaccine if they had HER2 IHC 1+/2+ disease (p = 0.04) [176]. When the follow-up was extended to 5 years, as the E75-specific immunity waned, the recurrences occurred more fre-quently. Therefore, an effective booster protocol was im-plemented on the patients who had failed to maintain a residual immunity [187]. No statistically significant differ-ences in DFS rates were registered in HER2 IHC3+ BC pa-tients. There are two current ongoing phase II trials trying to clarify the role of anti-HER2 vaccines in HER2-expressing BC patients, especially in relation to the con-comitant use of trastuzumab. One study is testing the E75 plus GM-CSF vaccine in combination with trastuzumab
in patients with high-risk, HER2 IHC 3+ BC [104] and the other one is evaluating the same combination with trastu-zumab in patients with HER2 low-expressing tumors (IHC 1+ or 2+) [120]. Moreover, a phase III clinical trial using E75 plus GM-CSF is currently ongoing for the adju-vant setting of patients with low to intermediate HER2 ex-pression BC [121].
GP2 vaccine A phase I trial explored the GP2 vaccine plus GM-CSF treatment feasibility in BC after standard adjuvant treatment. Eighteen node-negative, HER2 IHC 1+ to 3+ HLA-A2+ BC patients were vaccinated (6 of them were HER2 IHC 3+). Treatment was safe and elic-ited anti-HER2 specific immune response [186]. A sub-sequent phase II study was conducted to determine the efficacy for BC patients with high-risk, HER2 IHC 1+ to 3+ disease. A total of 180 patients were randomized to receive GP2 vaccine plus CSF (89 patients) or GM-CSF alone (91 patients). The 5-year DFS rate for the GP2 plus GM-CSF group vs. the GM-CSF only group, after a 34-month median follow-up, (per-treatment ana-lysis), was not significantly different, being respectively 94% and 85% (p = 0.43), and if only HER2 IHC3+/FISH+ patients were considered, 100% vs. 89%, respectively (p = 0.08). This trial suggested that vaccination may have clinical activity, particularly in patients with HER2 IHC 3+/FISH+ BC and that this benefit is to ascribed to the vaccine, with respect to the immune adjuvant alone [177]. Another phase Ib trial tested GP2 vaccine plus GM-CSF and trastuzumab in HER2+ (IHC 3+/FISH+) BC patients. Treatment was safe and stimulated a broad immunologic response supporting further investigation of combination strategies between anti-HER2 vaccines and trastuzumab [178].
AE37 vaccine AE37 vaccine plus GM-CSF was tested in
the adjuvant setting of BC patients. In a phase I trial, the vaccine showed to be safe and to be able to elicit HER-2/neu-specific immune responses [170]. A randomized phase II trial was conducted to determine efficacy of this vaccine for high-risk, HER2 IHC 1+ to 3+ BC patients. Two hundred ninety-eight patients were randomized to receive AE37 plus GM-CSF vaccine vs. GM-CSF alone. Among the HER2 IHC3+/FISH+ patients, 74 in the vac-cination group and 60 in the control group also received trastuzumab. The 5-year DFS rate after 25 months of median follow-up was 80.8% in the vaccinated group vs. 79.5% in the control patients (p = 0.7), when all patients were considered; 83.2% in vaccinated patients vs. 88.0% in controls, when HER2 IHC 3+/FISH+BC patients were considered (p = 0.45); 77.2% in the vaccinated ones vs. 65.7% in controls for the HER2 IHC 1+/2+ BC patients (p = 0.21); and 77.7% in vaccinated patients vs. 49.0% in control patients for TNBC (HER2 IHC 1+ to
2+/FISH-and HR-) (p = 0.12). In none of the subsets, a significant advantage in terms of DFS rate was seen. However, the study showed that the vaccine is safe and suggested that vaccination may have clinical benefit in patients with low HER2-expressing tumors, especially if TNBC [179]. The evidence available for anti-HER2 directed vaccines in the metastatic setting for HER2-expressing BC is lim-ited. Results from the most representative studies are summarized below.
Anti-recombinant HER2 protein (dHER2) vaccine A
phase I study assessed a recombinant HER2 protein (dHER2) vaccine combined with the immunostimulant AS15, in the adjuvant setting, in 61 trastuzumab-naive patients with HER2+ (IHC3+/FISH+) BC. The vaccine was safe and a specific immunological response was elic-ited, which increased with the dHER2 dose [186]. A paral-lel phase I/II study assessed the vaccine in 40 metastatic HER2 IHC3+/FISH+ BC patients, who received it as first-or second-line after a documented response to mainten-ance trastuzumab. The treatment was well tolerated. Both humoral and cellular immunity were activated, with one patient experiencing a complete response and another one a partial response. A stable disease was reported in 10 pa-tients [73]. A phase I trial showed that dHER2 vaccine plus AS15 could be given concomitantly with lapatinib in HER2 IHC3+/FISH+ mBC patients that were trastuzumab refractory. A total of 12 women were immunized while receiving lapatinib concurrently. The treatment was well tolerated, and anti-HER2 antibodies were induced in all patients, whereas HER2-specific T-cell response was detected in only 1 patient. There were no objective re-sponses. One patient remained free from disease progres-sion for 6 months. Median time to progresprogres-sion was 55 days, with the majority of patients progressing prior to the induction peak of anti-HER2 immune responses. How-ever, the 300-day OS rate was 92% [74].
Intracellular and extra-cellular domain anti-HER2 protein vaccines A phase I/II trial tested concurrent trastuzumab and ICD plus ECD peptide anti-HER2 vac-cination in patients with HER2 IHC 3+/FISH+ mBC, who had reached a complete remission or stable disease on trastuzumab treatment. Twenty-two patients on tras-tuzumab therapy were vaccinated. The treatment was well tolerated and immune response was significantly boosted and maintained. At a median follow-up of 36 months, mOS was not reached. A possible synergy be-tween trastuzumab and the vaccine was suggested [72].
Anti-MUC1 vaccines MUC1 is a glycoprotein
overex-pressed in BC. Its epitope was conjugated with a carrier protein to form a BC vaccine, which showed promising results in early-phase trials. However, a randomized
phase III trial that enrolled 1028 patients with mBC found no benefit from the vaccine in terms of time to progression (TTP) and OS. Sixteen percent of the pa-tients in the vaccination group and 14% in the control group had HER2+ BC [75].
Anti-WT1 vaccinesWilms’ tumor 1 antigen (WT1) can
be overexpressed in BC [188]. A phase I trial assessed a WT1 vaccine combined with AS15 in the neoadjuvant setting for WT1+ BC. Fourteen of the 62 vaccinated pa-tients had HER2+ BC and received concomitant trastuzu-mab chemotherapy. Nine of them had pCR, 4 had pPR, and 1 patient had no response. Treatment was well toler-ated, but humoral response could not be elicited, probably due to the co-administration of corticosteroids [180]. Anti-ganglioside vaccine N-glycolylsialic acid (NeuGc) containing gangliosides are overexpressed in many hu-man tumors and constitute potentially immunogenic molecules [189]. A phase I trial showed that a vaccine based on the NeuGc-GM3 ganglioside was safe and im-munogenic in advanced BC patients [190]. A phase III trial tested this vaccine in the adjuvant setting for BC patients. Of the 560 enrolled patients, 88 patients were randomly evaluated for immunological response, and only 22 of them had HER2+ BC. Seventeen of them ran-domly received the vaccine, while the other 5 received the placebo. The treatment was safe and high levels of anti-NeuGc-GM3 antibodies were detected, which was associated with a better clinical outcome [181].
Personalized mutanome vaccines Personalized
muta-nome vaccines are a new generation of promising anti-cancer vaccines that rely on the concept according to which tumor mutations are unique for individual pa-tients. The effective development of this therapeutical approach has become possible mainly thanks to the ad-vent of next-generation sequencing (NGS), which is as-sociated to dedicated bioinformatic tools in order to build a comprehensive map of tumor mutations (muta-nome) and a reliable prediction of epitope-binding to MHC molecules. The process to customize a mutanome cancer vaccine needs tumor tissue and healthy tissue from the patient. Subsequently, both tumor cells and normal cells are subjected to a comparative genomic se-quencing in order to find the somatic mutations and to exclude the germline mutations as possible epitopes. Once identified the somatic mutations, those with the highest likelihood to be recognized by the MHC molecules and those with a major clinical relevance are selected. After the process is completed, the epitopes generated are injected to the patient. To our knowledge, there are no published data of personalized mutanome vaccines in HER2-positive breast cancer patients. However, promising
results have been reported in other cancer types. A phase I trial evaluated the safety and efficacy of personalized vac-cines in untreated stage III and IV melanoma patients. Of six vaccinated patients, 4 were disease free at 25 months of follow up while 2 patients who progressed to the vaccine treatment received an anti PD1 therapy obtaining a radiological complete response. The immunological as-says showed an important immune system activation against the neoepitopes which involved both CD4+ and CD8+ cells [191]. Another study conducted in 13 pa-tients with stage III and IV melanoma tested a RNA-based poly neo-epitope showing similar outcomes: all patients developed a T cell response to the treatment and the rate of metastatic events were significantly re-duced after the start of vaccination (p < 0.0001) which translated into a better PFS [192].
Gene-based vaccines
The gene-based vaccine formulations use DNA structures of viruses or plasmids to deliver DNA sequences coding for TAAs. They are safe and can potentially elicit both humoral and cell-mediated immune response [193,194]. Viral vector-based vaccines
Panvac Viral vector-based vaccines have been used for TAAs such as HER2, p53, and MUC1, mostly in clinical trials involving metastatic BC patients [195]. PANVAC is a cancer vaccine therapy delivered through two viral vectors, which include transgenes for MUC-1, CEA, and for three human T cell costimulatory molecules [196]. This vaccine was safe in mBC patients and showed activ-ity [197]. A phase II trial used docetaxel with or without PANVAC plus GM-CSF in mBC patients. Twenty-five patients received docetaxel and the vaccine, while 23 re-ceived standard docetaxel alone. Six HER2+ patients were included, 3 for each arm. The combination treat-ment of docetaxel with PANVAC resulted in a longer progression-free survival (PFS) compared to docetaxel treatment alone (7.9 vs. 3.9 months) (p = 0.09), but the number of HER2+ BC patients was too scarce to draw any firm conclusion [76].
Plasmid vector-based vaccines
Plasmid DNA can induce antibody responses to viral and non-viral antigens [198].
Anti-HER2 DNA vaccine A pilot clinical trial used a
plasmid vaccination with DNA encoding for HER2 pro-tein plus low doses of GM-CSF and interleukin-2 (IL-2), in patients with HER2+ mBC, who were also treated with trastuzumab. Vaccination was well tolerated and could induce long-lasting cellular and humoral immune responses against HER2 protein [77].
Anti-mammaglobin-A DNA vaccine Mammaglobin-A
(MAM-A) is overexpressed in 50% of HER2+ BCs [199]. A phase I study investigated a MAM-A plasmid DNA vaccine in patients with stable mBC. A total of 14 patients were vaccinated and compared to controls. Vaccination was safe, yielded a significant increase in CD8+ T cell re-sponse, and improved PFS rate at 6 months follow-up (53% vs. 33%; p = 0.011). Three patients of the 14 vacci-nated had HER2+ BC vs. 1 in the control group [78]. Whole-cell vaccines
These vaccine formulations can use tumor cells, DCs, or T lymphocytes [79].
Tumor cell-based vaccines
Vaccine strategies based on direct application of whole tumor cells or cell extracts are polyvalent immunization strategies [200], which have shown a relatively poor immunogenic potential [201]. They are prepared by irradiating allogeneic or autologous cancer cells or cell lysates [202, 203] and their immunogenicity could be increased by engineering tumor cell lines to
se-crete GM-CSF [204, 205] and/or by combination with
chemotherapy [206, 207].
A phase I study investigated a treatment with cyclo-phosphamide (CY), doxorubicin (DOX), and an anti-HER2 GM-CSF secreting allogenic BC cell line vaccine. This study enrolled 28 patients with stable mBC, of whom only 1 had HER2+ BC. The treatment was safe and bioactive when given alone or after low-dose CY and DOX [80]. Another phase I study treated mBC pa-tients with a vaccine of an allogeneic HER2+ BC cell line genetically modified to express B7-1 costimulatory pro-tein, in association with GM-CSF. Thirty pretreated women were vaccinated, of whom 12 were with a HER2+ disease. Prolonged stable disease was observed in 4 patients but no objective response was registered. Treatment was well tolerated [81].
Another study investigated the feasibility of a treat-ment with cyclophosphamide, trastuzumab, and a vac-cine of an allogeneic HER2+ BC GM-CSF secreting cell line in 20 patients with HER2+ mBC. The treatment was safe and yielded considerable clinical benefits in terms of OR, PFS, and OS [82].
Dendritic cell-based vaccines
It is one of the most relevant approaches in the field of BC vaccine development. Autologous DCs are loaded in vitro with cancer antigens using different techniques such as facilitating antigen presentation with synthetic-ally developed peptides, antigens derived from killed autologous/allogeneic BC cells [208] or antigens isolated from cancer stem cells [209]. TAAs presence on DC sur-face can also be obtained by physically fusing them with
cancer cell lines [210, 211]. Her2 protein antigens have been widely used in DC vaccines. A phase I study inves-tigated a DC vaccine loaded with a HER2 antigen called Lapuleucel-T to treat patients with HER2+ mBC. APCs were activated in vitro with a recombinant fusion pro-tein consisting of ICD and ECD sequences of HER2 linked to GM-CSF. Eighteen patients were treated with a resulting good tolerability. Significant cell-mediated anti-HER2 immune response was induced. Clinical benefit in terms of tumor response was recorded [83]. Another phase I study investigated an anti-HER2 DC-based vac-cination on 13 patients with HER2 IHC3+ DCIS BC in the neoadjuvant setting showing specific activation of T cells, accumulation of T and B lymphocytes in the breast, and induction of tumor-lytic antibodies [182]. Subsequently, another group of study conducted two phase I clinical trials in the neoadjuvant setting for pa-tients with HER2+ DCIS BC using a DC-based vaccine pulsed with a mixture of ICD and ECD HER2 peptides. In the first study, 27 HER2 IHC3+ BC patients were vaccinated. The first analysis showed the induction of a potent type I immunity, suggesting potential activity at a clinical level [183]. A subsequent analysis showed that the vaccine could eliminate HER2 expression in 50% of patients [212]. The second trial treated 54 patients, of whom one third had HER2 IHC2+ and two thirds had HER2 IHC3+. The vaccination was safe and immuno-genic. For all the patients who achieved pCR, an anti-HER2 T-cell response was recorded in the sentinel lymph node [184]. A previous small study tested an anti-HER2 DC-based vaccine in the adjuvant setting for 7 women with high risk invasive HER2+ (IHC2+ to 3+/ FISH+) BC, which had undergone standard treatment (± trastuzumab). At a 5-year follow-up, 6 patients had measurable anti-HER2 antibodies and all patients were alive [79]. A phase II trial testing efficacy in HER2+ mBC patients of vinorelbine plus an anti-HER2 DC vac-cine plus tratuzumab in association with GM-CSF is on-going [105] (see also Tables3and4).
Autologous T cell-based vaccine
There are very few data on the treatment of HER2+ BC with vaccines based on autologous T cells.
A small pilot study on only one patient with HER2+ mBC tested an adoptive transfer of autologous HER2-specific T-lymphocytes, which resulted unable to pene-trate into the solid metastases. However, disseminated tumor cells in the bone marrow disappeared after the completion of the treatment [213]. Another phase I trial investigated the feasibility of a vaccine with HER2 primed autologous T cells in 7 patients with HER2+ treatment refractory mBC. The treatment was feasible and clinical responses were observed in 43% of patients. Lower number of T-regulatory (T-reg) cells in peripheral
blood prior to infusion (p < 0.001), higher level of HER2 specific T-cells in vivo (p = 0.030), and development of diverse clonal T-cell populations (p < 0.001) were posi-tively associated with response [84]. A further step in the autologous T cell vaccine strategy has started to be explored recently by using T lymphocytes genetically engineered to express chimeric antigen receptors (CAR-Ts). CAR-T cell therapy is currently being investigated in a phase I study enrolling patients with relapsed or re-fractory HER2+ solid tumors (breast cancer, ovarian cancer, lung cancer, gastric cancer, colorectal cancer, gli-oma, pancreatic cancer) [106].
Currently ongoing trials of immunotherapy in HER2+ BC are listed in Tables3and4.
Discussion
The overall cumulated evidence analyzed in this review showed that the modulation of the immune system for the treatment of HER2+ BC is a promising path. Trastu-zumab’s passive ability to exploit immune response for its action can be enhanced and partially redirected in an advantageous way by strategies that refine its structure or by combining it with other agents in such a way that synergy is yielded in activating immune response. Ap-proaches that addressed experimentally the modification of trastuzumab structure include the conceptualization of T-DM1, which facilitates DC’s functions; margetuxi-mab, a mAb that increases the ACCD; and the bispecific mAb MCLA-128, capable of enhancing both ACCD and the anti-trophic effect. Studies showed that the ACCD effect of trastuzumab can also be amplified by combin-ing it with lapatinib. Other bispecific mAbs were engi-neered with the intent of promoting the encounter between HER2+ BC cells and lymphocytes, while facili-tating the activation of these latter. Such mAbs include formulations able to bind on one side the HER2, and on the other side the T cell’s and/or NK cell’s specific re-ceptors or costimulatory molecules such as CD16, CD 137, and CD3. These strategies showed efficacy in pre-clinical and early pre-clinical trials, some of which were on-going at the time this review was thought and conceived. Additional approaches are being investigated mostly in preclinical studies. In vitro studies showed that nano-body agents can selectively redirect pre-existent anti-bodies towards HER2+ BC cells inducing ACCD. Other studies suggest a possible advantage in terms of an in-crease in TILs and immune response when combining anti-HER2 therapy functionally and/or structurally with cytokines such as IL-2 or IFN-γ. All these modalities offer a wide landscape where the immune-mediated mechanism of anti-HER2 agents can be further ex-tended. Moreover, on a parallel path, the generic activity of trastuzumab can also be enhanced using other modal-ities, such as association with chemotherapy, labeling it
Table 3 Ongoing trials with immunotherapy in HER2 + m e tastatic breast cancer patients Drug Phase Setting Status Type of intervention Mechanism of action NCT02492711 [ 91 ] Margetuximab III Metastatic Completed HER2-block enhancer Increased ADCC effect NCT03330561 [ 96 ] PRS-343 I Metastatic Recruiting Immune costimulatory T-Cell activation NCT03650348 [ 97 ] PRS-343+ Atezolizumab I Metastatic Recruiting Immune costimulatory+ Immune check point inhibitor T-Cell activation+ Anti PD-L1 NCT02829372 [ 98 ] NA I Metastatic Recruiting Immune costimulatory (anti HER2/ CD-3 antibody) ADCC + T-Cell activation NCT02627274 [ 100 ] RO6874281 I Metastatic Recruiting Immune costimulatory ADCC + T-Cell activation DIAmOND [ 101 ]
Durvalumab+ Tremelimumab+ Trastuzumab
II Metastatic Recruiting Immune check point inhibitor Anti PD-L1 + Anti-CTLA-4 NCT03414658 [ 102 ] Avelumab + Utomilumab + Vinroelbine+ Trastuzumab II Metastatic Recruiting Immune costimulatory+ Immune check point inhibitor T-Cell activation+ Anti PD-1 NCT03199885 [ 103 ] Paclitaxel + Trastuzumab + Pertuzumab + Atezolizumab III Metastatic Recruiting Immune check point inhibitor Anti PD-L1 NCT02297698 [ 104 ] Nelipepimut+ Trastuzumab II Metastatic Active, not recruiting Vaccine Peptide-based vaccine (E75) NCT00266110 [ 105 ] therapeutic autologous dendritic cells+ Trastuzumb + Chemotherapy II Metastatic Completed Vaccine DC vaccine NCT02713984 [ 106 ] Anti-HER2 CAR-T I Metastatic Recruiting Vaccine CAR-T cells NCT03125928 [ 107 ] Pertuzumab + Trastuzumab + Atezolizumab IIA Metastatic Recruiting Immune check point inhibitor Anti PD-L1 NCT02605915 [ 108 ] Atezolizumab+ Pertuzumab + Trastuzumab + or Atezolizumab+TDM-1 IB Early and metastatic Active, not recruiting Immune check point inhibitor Anti PD-L1 NCT03417544 [ 109 ] Atezolizumab+Pertuzumab + Trastuzumab + II † Metastatic Recruiting Immune check point inhibitor Anti PD-L1 NCT00343109 [ 110 ] NA II Metastatic Active, not recruiting Vaccine ICD peptide-based vaccine NCT02547961 [ 111 ] NA I/II Metastatic Completed Vaccine CAR-T cells NCT03364348 [ 112 ] Utomilumab + TDM-1 or trastuzumab I Metastatic Recruiting Immune costimulatory T-Cell activation NCT01922921 [ 113 ] I/II Metastatic Active, not recruiting Vaccine ICD peptide-based vaccine NCT03032107 [ 114 ] Pembrolizumab+ TDM-1 I Metastatic Recruiting Immune checkpoint inhibitor Anti PD-1 NCT00436254 [ 115 ] NA I Early and metastatic Active, not recruiting Vaccine pNGVL3-hICD vaccine NCT00194714 [ 116 ] NA I/II Metastatic Active, not recruiting Vaccine Her2+ peptide vaccine NCT03740256 [ 117 ] NA I Metastatic Not yet recruiting Vaccine Adenovirus-specific cytotoxic T lymphocytes (HER2-AdVST), in combination with intra-tumor injection of CAdVEC, an oncolytic adenovirus mAbdirected against fibroblast activation protein-alpha (FAP) linked to an engineered, variant form of interleukin-2 (IL-2v) †Only HER2+ patients with brain metastasis Abbreviations: ADCC antibody-dependent cytotoxic cell, CAR chimeric antigen receptor, DC dendritic cell, ICD intracellular domain, mAb monoclonal antibody, NA not available
Table 4 Ongoing trials with immunotherapy in HER2 + breast cancer patients, early settin g D rug Phas e Setting Stat us Type of interve ntion Mech anism of action NCT0 35955 92 [ 118 ] Atez olizumab + Pe rtuzum ab+ Tra stuzum ab+ Ch emotherap y III Ear ly Re cruiting Immun e chec k point inhib itor Anti PD-L1 NCT0 37268 79 [ 119 ] Atez olizumab + Pe rtuzum ab+ Tra stuzum ab+ Ch emotherap y III Ear ly Re cruiting Immun e chec k point inhib itor Anti PD-L1 NCT0 15700 36 [ 120 ] Ne lipepim ut+ Tra stuzum ab II Ear ly ‡ Comp leted Vaccine Peptid e-based vacci ne (E7 5) NCT0 14792 44 [ 121 ] Ne lipepim ut+ Sag ramostim II Ear ly ‡ Comp leted Vaccine Peptid e-based vacci ne (E7 5) NCT0 26059 15 [ 108 ] Atez olizumab + Pe rtuzum ab + Tra stuzum ab + or Atez olizumab +TDM -1 IB Ear ly an d meta static Ac tive, not recru iting Immun e chec k point inhib itor Anti PD-L1 NCT0 33219 81 [ 122 ] M C LA-128 II Ear ly Re cruiting HER2-bl ock enhanc er ADCC stim ulation against HER2 /HER3 recept ors NCT0 37471 20 [ 123 ] P e mbrolizumab+ Pacli taxel + Pertuz umab + Tra stuzum ab II Ear ly Re cruiting Immun e chec k point inhib itor Anti PD-1 NCT0 13553 93 [ 124 ] N A I/II Ear ly Ac tive, notrec ruiting Vaccine Peptid e vacci ne NCT0 04362 54 [ 115 ] NA I Ear ly an d Met astat ic Ac tive, not recru iting Vaccine pNGVL3 -hICD vaccine NCT0 37429 86 [ 125 ] Ni volumab + docetaxe l+ Pe rtuzum ab+ Tra stuzum ab II Ear ly Not yet recruit ing Immun e chec kpoint inh ibitor Anti PD-1 NCT0 33875 53 [ 126 ] NA I Ear ly Re cruiting Vaccine DC Vacc ine NCT0 36308 09 [ 127 ] NA II Ear ly Re cruiting Vaccine DC vac cine NCT0 36202 01 [ 128 ] N A I Ear ly Re cruiting Immun e chec kpoint inh ibitor Anti PD-L1/TGF-Be ta TRAP . NCT0 38201 41 [ 129 ] D urvalumab + Tra stuzum ab+ Pe rtuzum ab II* Ear ly Not yet recruit ing Immun e chec kpoint inh ibitor Anti PD-L1 ‡Only patients with HER2 1+ and 2+ expressing tumors *Only HER2-enriched patients Abbreviations: ADCC antibody-dependent cytotoxic cell, CAR chimeric antigen receptor, DC dendritic cell, ICD intracellular domain, mAb monoclonal antibody, NA not available
